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Temperature-Dependent Structural Variability of Prion Protein Amyloid Fibrils

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Temperature-Dependent Structural Variability of Prion Protein Amyloid Fibrils International Journal of Molecular Sciences Article Temperature-Dependent Structural Variability of Prion Protein Amyloid Fibrils Mantas Ziaunys, Andrius Sakalauskas , Kamile Mikalauskaite, Ruta Snieckute and Vytautas Smirnovas * Life Sciences Center, Institute of Biotechnology, Vilnius University, LT-10257 Vilnius, Lithuania; [email protected] (M.Z.); [email protected] (A.S.); [email protected] (K.M.); [email protected] (R.S.) * Correspondence: [email protected] Abstract: Prion protein aggregation into amyloid fibrils is associated with the onset and progression of prion diseases—a group of neurodegenerative amyloidoses. The process of such aggregate formation is still not fully understood, especially regarding their polymorphism, an event where the same type of protein forms multiple, conformationally and morphologically distinct structures. Considering that such structural variations can greatly complicate the search for potential antiamyloid compounds, either by having specific propagation properties or stability, it is important to better understand this aggregation event. We have recently reported the ability of prion protein fibrils to obtain at least two distinct conformations under identical conditions, which raised the question if this occurrence is tied to only certain environmental conditions. In this work, we examined a large sample size of prion protein aggregation reactions under a range of temperatures and analyzed the resulting fibril dye-binding, secondary structure and morphological properties. We show that all temperature conditions lead to the formation of more than one fibril type and that this variability Citation: Ziaunys, M.; Sakalauskas, may depend on the state of the initial prion protein molecules. A.; Mikalauskaite, K.; Snieckute, R.; Smirnovas, V. Temperature- Keywords: amyloids; prion proteins; protein aggregation; fibril structure Dependent Structural Variability of Prion Protein Amyloid Fibrils. Int. J. Mol. Sci. 2021, 22, 5075. https:// doi.org/10.3390/ijms22105075 1. Introduction Amyloidogenic protein aggregation into insoluble, beta-sheet rich fibrils is linked with Academic Editor: the onset of several neurodegenerative disorders, such as Alzheimer’s, Parkinson’s or prion Vladimir N. Uversky diseases [1,2]. The way these structures form and propagate is still not fully understood, as evidence for new aggregation mechanisms or fibril structural features [3–5] keeps appearing Received: 20 April 2021 on a regular basis. This lack of crucial information is likely one of the factors that has led Accepted: 9 May 2021 Published: 11 May 2021 to countless failed clinical trials [6] and to only a handful of effective, disease-modifying drugs [7]. Considering that amyloid diseases affect millions of people worldwide and the Publisher’s Note: MDPI stays neutral number is expected to continuously increase [8,9], it is of vital importance to gain a better with regard to jurisdictional claims in understanding of the intricacies of amyloid aggregation. published maps and institutional affil- One of the more interesting aspects of amyloid aggregation is the ability of a single iations. type of protein to associate into multiple conformationally-distinct fibrils [10]. Such a phenomenon was observed with prion proteins, both in vivo and in vitro [11–14] and later– with other amyloidogenic proteins, such as amyloid beta [15], alpha-synuclein [16,17] and insulin [18,19]. These distinct conformation fibrils possess specific replication rates [20,21], morphologies [22], secondary structures [16,23] and stabilities [24]. In addition, some of Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. these parameters are either codependent or change even after fibrils have been formed. This article is an open access article It has been shown using computational methods that there is a correlation between the distributed under the terms and rate of self-replication and fibril symmetry, and stability [25,26]. It was also observed conditions of the Creative Commons that oligomeric or protofibrillar intermediate aggregates gain mechanical stability when Attribution (CC BY) license (https:// converting to fully formed fibrils [27]. creativecommons.org/licenses/by/ Such a variation in their properties is likely one of the main aspects why potential 4.0/). drug candidates appear effective under a certain set of conditions, while being completely Int. J. Mol. Sci. 2021, 22, 5075. https://doi.org/10.3390/ijms22105075 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 5075 2 of 16 inefficient in others [28]. It has been observed in multiple studies that the conformation of resulting fibrils depends highly on the environmental conditions, under which the protein is aggregated [29,30]. These conditions include temperature [31], agitation [32], pH [18], ionic strength [33], denaturant concentration [14] and protein concentration [34]. In our recent study, we have also shown that prion proteins are capable of forming two distinct conformation fibrils under identical conditions [23]. This hints at a possibility that primary nucleation, a process during which the initial aggregation centers from, may randomly generate distinct nuclei, only some of which can further propagate under the given environmental conditions. Determining conformational differences between amyloid fibrils is typically done by evaluating their morphological features [35], such as aggregate height, width, length and periodicity patterns, and by acquiring information about their secondary structure [36]. The methods used for this, namely atomic force microscopy (AFM) [35] and Fourier-transform infrared spectroscopy (FTIR) [37], are difficult to apply in situations, where a large number of samples have to be differentiated. In recent years, both high-throughput screening platforms [38] and computer-aided molecule design systems [39] have advanced enough to identify neurodegenerative-disease related protein–ligand interactions and possible mechanisms of fibril formation. However, since protein aggregation is still not fully understood, such methods are not ideal for determining the highly complex structure of fully formed aggregates. As an alternative, the selective and conformation-specific binding of a fluorescent probe, thioflavin-T (ThT) [23,40,41], may be used to detect different types of aggregates. Such a method was applied in our aforementioned study with prion proteins and it was efficient at identifying different types of insulin fibrils as well [40]. Applying this type of initial screening would allow to sort out fibril samples with distinct conformations in a much larger scale assay. Prion protein aggregation experiments are conducted under a variety of conditions in vitro, ranging from ambient temperature [42] to well-above physiological tempera- tures [43]. In order to determine if this environmental factor has an effect on prion protein fibril variability, we tracked the aggregation of a mouse prion protein under a range of different temperatures, using a large identical sample size to evaluate their seemingly random, conformational variations. To achieve complete fibrillization in a reasonable time frame, vigorous, fragmentation-inducing agitation was also used for every temperature condition. A ThT-assay was then employed as an initial means of sorting the distinct fibril types, which were then examined using FTIR and AFM. 2. Results A large sample size of mouse prion protein fragment (89–230) was aggregated un- der a range of temperatures (from 25 to 65 ◦C) with all other conditions, such as buffer solution, protein concentration and volume remaining identical. Plotting the lag time (tlag) dependence on aggregation temperature (Figure1A) revealed a discontinuity in the linear trend at 45 ◦C. This is caused by the protein transitioning from being in mostly folded states (at temperatures below 45 ◦C) and mostly unfolded (above 45 ◦C) [43]. This is further supported by visualizing the data in an Arrhenius plot (Figure1B), where the activation of nuclei formation was (78.7 ± 7.0) kJ/mol at low and (30.0 ± 5.0) kJ/mol at high temperatures. This transition temperature is in line with previously reported data, [43] where a shift in activation energy was also observed. Int. J. Mol. Sci. 2021, 22, x 3 of 18 way analysis of variance (ANOVA) statistical analysis of the data (n = 90 for each case) using a Bonferroni means comparison (significance level–0.01) revealed that there was a difference between the 35–45 °C sample and 50–60 °C sample fluorescence intensity dis- tributions, where 35–45 °C fibril-bound ThT possesses a considerably larger average sig- nal intensity. Interestingly, there was no significant difference between the lowest and highest temperature sample sets (significance level–0.01), apart from the 25 °C samples having a greater standard deviation due to the existence of a few high fluorescence inten- sity samples. Both of these aspects could be explained by a larger fibril concentration or the formation of superstructural fibril assemblies, however, that is not the case. Each sam- ple contained an identical concentration of prion protein and their aggregation kinetic curves reached a plateau, indicating a finished fibrillization process. Afterwards, each sample was diluted with the reaction buffer (containing 100 µM ThT) and sonicated, which removes the possibility of large aggregate structures entrapping the dye molecules Int. J. Mol. Sci. 2021, 22, 5075 [44] or certain samples having
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